Presently, surface expression of proteins has been investigated in unicellular organisms such as bacteriophage, bacteria, yeast and the like to produce new vaccines, to perform screening of various antigens and antibodies and to localize useful enzymes onto a cell surface.
Surface expression of proteins has been tried at first to express antigenic peptides onto a cell surface for stable production of vaccines. Hitherto, in order to produce vaccines pathogenic bacteria were mutated arbitrarily and screened for selecting safe bacteria which can induce immunization consistently. However, the screening method has a disadvantage of losing the antigenic activities when the vaccines are administered orally into human body and animal body. Thus, many investigations have been performed to overcome the disadvantage.
First, surface expression has been performed by the process, wherein gene segments of cell surface protein were exploited in gram-negative bacteria, ligated to genes of antigenic polypeptides and used to transform proper bacteria hosts for effective production of fusion proteins. The fusion proteins prepared by the process can work as effective antigens since they are extruded onto a cell surface stably. Especially in gram-negative bacteria, outer member lipopolysaccharides (LPS) increase antigenicity of proteins expressed onto a cell surface.
To express proteins onto a cell surface, a secretion signal is necessary within primary sequences of proteins, which helps foreign proteins produced intracellularly to pass through a cell membrane. Especially in gram-negative bacteria, proteins should pass through the inner cell membrane and periplasmic space, localize onto the outer cell membrane stably and be extruded. For example, surface proteins, specific enzymes and toxin proteins have a secretion signal and a targeting signal localizing the proteins onto the cell surface. Practically, foreign proteins can be expressed on the cell surface successfully by using such a secretion signal, a targeting signal and the like combined with proper promoters.
Up to now, surface proteins present in gram-negative bacteria have been utilized mainly to produce foreign polypeptides which are necessary onto the cell surface. There are 4 kinds of proteins used for the cell surface expression, such as outer cell membrane protein, lipoprotein, secretion protein and cell surface structure protein. As outer cell membrane proteins, Lam B, Pho E, Omp A and the like have been utilized for the surface expression. In these cases, however, sizes of proteins expressed onto the cell surface are limited since the proteins should be inserted into loops extruded from the cell surface. In addition, the C- and N-termini of the foreign protein should be close to each other 3- dimensionally. Thus, when the distance between the two termini is long the C- and N-termini should be joined.
Practically, if Lam B or Pho E is utilized to insert foreign polypeptides comprising more than 50-60 amino acids, membrane proteins cannot be produced stably due to structural imitation [Charbit, et al., J. Immuol., 139: 1658-1664 (1987); Agterberg, et al., Vaccines, 8: 85-91 (1990)]. To overcome the structural limitation, a part of the Omp A protein which contains a miniumum targeting sequence localizing foreign proteins onto the outer cell membrane is used, although the whole Omp A protein was also used to insert foreign proteins into the extruded loop. By the process described about, .beta.- lactamase was expressed onto the cell surface by fusing the C-termainal targeting sequence of Omp A protein. In this case, Omp A fragment helped proteins to be expressed and bound onto the cell surface and a signal sequence of E. coli lipoprotein, Lpp, which was fused to N-terminus of Omp A helped the protein to be localized onto outer cell membrane [Francisco, et al., Proc. Natl. Acad. Sci. USA, 489: 2713-2727 (1992)].
Therefore, the cell surface expression using outer membrane proteins should be performed in bacteria by the process, wherein a selected outer membrane protein fused with foreign proteins in the level of genes, is used to induce biosynthesis of fusion proteins, passes through the inner membrane stably and maintains outer membrane binding of foreign proteins. Thus, outer membrane proteins selected as a surface anchoring motif must satisfy requirements described below. The outer membrane proteins should i) have a secretion signal by which a fusion protein can pass through the inner cell membrane, ii) have a targeting signal by which the protein can bind onto the outer cell membrane, iii) be expressed massively on the cell surface and iv) be expressed stably regardless of protein size. However, any cell surface anchoring motif satisfying all the requirements has not been yet developed and only supplemented the above disadvantages.
Lipoproteins, as a surface protein, have also been used for the surface expression. Particularly, E. coli lipoproteins can pass through the inner cell membrane due to a secretion signal at the N-terminus and can localize directly to outer or inner membrane lipids by covalent bonding due to terminal L-cysteine. In addition, since a major lipoprotein, Lpp, binds to the outer cell membrane at the N-terminus and to the cell layer, peptidoglycan (PG), at the C-terminus, a foreign protein joined with outer membrane Omp A fragment can be secreted stably onto the outer cell membrane for the surface expression [Francisco, et al., Proc. Natl. Acad. Sci. USA, 489: 2713-2727 (1992)]. By using the characteristics of lipoproteins, another lipoprotein, Tra T, has been exploited to express peptides such as Poliovirus C3 epitope and the like onto the cell surface [Felici, et al., J. Mol. Biol., 222: 301-310 (1991)]. In addition, a peptiodoglycan-associated lipoprotein (PAL) of which the function is not known exactly has also been exploited to express recombinant antibodies on the cell surface [Fuchs, et al., Bio/Technology, 9: 1369-1372 (1991)]. In this case, PAL was joined with peptiodoglycan at the C-terminus and with the recombinant antibody at the N-terminus to express the fusion protein on the cell surface.
Secretion proteins, as a surface protein passing through the outer cell membrane, have been used for the surface expression. However, in gram-negative bacteria secretion proteins are not well-developed and only some secretion proteins participate in passing through the outer cell membrane by the proteins helping the specific secretion process. For example, a pullulanase from Klebsiella sp. is replaced with a lipid component at the N-terminus, binds to the outer cell membrane and is secreted into cell media. Kornacker et al., tried to express .beta.-lactamase on the cell surface by using the N-terminal fragment of pullulanase. Unfortunately, the pullulanase-.beta.-lactamase fusion protein bound to the cell surface instantly and was secreted into cell media. Also, alkaline phosphatase, a periplasmic space protein, was attempted to be used by the above process. But the alkaline prosphatase was not expressed stably onto the cell surface since more than 14 proteins are necessary in the secretion process [Kornacker, et al., Mol. Microl., 4: 1101-1109 (1990)].
Ig A protease is derived from a pathogenic microorganism, Neisseria and has a unique secretion mechanism. The C-terminal .beta.-fragment of Ig A protease has a signal by which the N-terminal protease can be localized onto the outer cell membrane. After the protease reaches the outer cell membrane to be extruded on the cell surface, the extruded protease is secreted into cell media by auto-hydrolysis. By using the .beta.-fragment of the Ig A protease, Klauser et al. also expressed 12 kDa chloera toxin B subunit stably onto the cell surface [Klauser, et al., EMBO J., 9: 1991-1999 (1990)]. However, secretion of the fusion protein was inhibited since a protein folding is induced in the periplasmic space during the secretion process.
In addition, other cell surface structures which are present on cell surface, such as flagella, pili, fimbriae and the like, can be used for the surface expression. By using flagellin, a structural subunit of flagella, respective chlolela toxin B subunit and other peptides derived from Hepatitis B Virus were expressed stably and these peptides could bind with the respective antibody intensively [Newton, et al., Science, 244: 70-72 (1989)]. By using fimbrin, a structural protein of fimbriae which works on cell surface like threads, foreign proteins were also expressed. As a result, only small peptides have been expressed successfully [Hedegaard, et al., Gene, 85: 115-124 (1989)].
In addition to the surface proteins of gram-negative bacteria, those of gram-positive bacteria has been attempted to express foreign proteins on cell surface recently [Samuelson, et al., J Bacteriol., 177: 1470-1476 (1995)]. Also, a surface anchoring motif passing through the inner cell membrane and binding to the cell membrane is necessary. Practically a secretion signal of lipase derived from Staphylococcus hyicus and a membrane-bound matrix of protein A derived from Staphylococcus aureus sere used to express foreign proteins onto the cell surface. As a result, a malaria blood state antigen comprising 80 amino acids and an albumin binding protein derived from G protein of Streptococcus were expressed on the cell surface of gram-positive bacteria efficiently.
In investigating the surface expression in gram-negative and gram-positive bacteria as described above, useful systems for the protein expression have been developed a lot. For recent 3 years, the systems have been filed and registered as patent rights in USA, Europe, Japan and the like. In particular, 5 cases using the outer cell membrane of gram-negative bacteria were registered Also, 1 case using a pilus, a cell surface structure, and 1 case using a cell surface lipoprotein were registered.
The inventors have exploited ice nucleation protein (INP), a surface protein derived from Pseudomonas syringae KCTC 1832, as a new surface anchoring motif and developed new surface anchoring vectors containing INP for expressing foreign proteins efficiently on a cell surface, a method for preparation of foreign proteins onto a cell surface and use thereof.